Solid State NMR Structural Studies of Antimicrobial Peptides LPcin Analogs with Enhanced Activities

Abstract

The availability of antibiotics has allowed for the successful treatment of many bacterial infections as well as the ability to perform invasive medical procedures including surgery and chemotherapy. However, their wide use has led to pathogens’ increased drug resistance and the need to find novel classes of antimicrobial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113-135 region of component-3 of proteose-peptone. LPcin is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering in our laboratory. These three LPcin analogs show better antibioitic activities than wild-type LPcin and no toxicity at all.In order to understand the structural correlation between LPcin analogs structure and antimicrobial activity under the membrane environments, we tried to express and purify as large as amounts of LPcin and three different LPcin analogs. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with biophysical techniques like Ni-affinity chromatography, dialysis, centrifuge, chemical cleavage, and reversed-phase semiprep HPLC. In here, we will present the optimizing processes with high-yield expression and purification of three LPcin analogs and solid- state NMR structural studies to figure out antibacterial killing mechanisms.